title: High-throughput knock-in coupling gene targeting with the HPRT minigene and Cre-mediated recombination.
abstract: Single nucleotide polymorphisms (SNPs) may influence protein function possibly contributing to phenotype; yet, for most SNPs their potential influence is unknown. Here, we present a technique in mouse embryonic stem cells that enables high-throughput knock-in (the placement of coding sequences adjacent to a specific endogenous promoter). Our methodology utilizes gene targeting with a combination of two selection cassettes (SAbetageo and the HPRT minigene) along with site-specific recombinases (Cre/loxP and FLP/FRT) to efficiently introduce multiple DNA sequences, including enhanced green fluorescent protein (eGFP), adjacent to the DNA topoisomerase 3beta (Top3beta) promoter. This technology enables rapid and efficient introduction of DNA sequences to a specific location and advances high-throughput analysis of many SNPs with control for expression and genetic background.
authors.affiliation: The Department of Molecular Medicine, The Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA.
authors.forename: Tae Moon
authors.initials: TM
authors.lastname: Kim
authors.forename: Yong Jun
authors.initials: YJ
authors.lastname: Choi
authors.forename: Jun Ho
authors.initials: JH
authors.lastname: Ko
authors.forename: Paul
authors.initials: P
authors.lastname: Hasty
mesh_terms: D000818:Animals; D002678:Chimera / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism; D055879:Gene Knock-In Techniques; D018390:Gene Targeting / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D019426:Integrases / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000502:physiology; D011995:Recombination, Genetic
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase; C045073:Cre recombinase; D019426:Integrases